The Mechanism Of MAPK Signaling Pathway In The Electrotaxis Migration Of Soil Amoeba Dictyostelium Discoideum

Dictyostelium discoideum is one of the soil microorganism that exists in nature.It often feeds on bacteria and yeasts in the soil,and it sows and harvests "food" in an "agricultural" behavior similar to humans.When environmental stress(such as chemical,physical factor stress,etc.)appears,it will start a unique lifestyle,that is,a transition from a single-cell free lifestyle to a multicellular organism that forms a sporangia structure.During multicellular development,a special bioelectric field is formed and guides the directional migration of cells and the development of multicellular organisms.Many eukaryotic cells are capable of sensing environmental signals,including light,chemicals,mechanical forces,electric fields and temperature.After receiving foreign signals,cells can rearrange their polarity,adhesion and cytoskeleton by activating signal transduction pathways or regulating gene expression,thus completing cell migration.Directed cell migration is an important life process of single-celled organisms and the basis for the establishment and maintenance of unique organizational structure.Cell directed migration plays many key roles in embryo development,angiogenesis,immune response,wound healing and tumor metastasis in multicellular organisms.Electric field directed cell migration behavior is called cell electrotactic migration.Previously,a variety of cells,such as corneal epithelial cells,keratinocytes,and endothelial cells,have been proved to be able to respond to electrical signals outside the cell and undergo directional migration.Most cells migrated to the negative electrode in uniform electric field.And a few cells can migrate to the positive terminal.The study of cell electrotaxis shows that is not a simple electrophoretic phenomenon and different from cell chemotaxis.However,there are still many problems to be explored in the study of the molecular mechanism of cell electrotaxis.Among them,Ras/MAPK/ERK pathway has attracted the most attention in recent years,which is not only highly correlated with cell migration,but also clearly correlated with ERK phosphorylation of downstream factor of MAPKs pathway and cell electrotaxis.However,whether its upstream signal is related to cell electrotaxis remains unclear.In order to further explore the role of MAPKs signaling pathway in cell electrotaxis,the model organism Dictyostelium discoideum was used as the study object.Multi-dimensional analysis of Ras/MAPK/ERK pathway under exogenous electric field stimulation was performed by Time-lapse living cell micrography,Western blot,transcriptomics,protein modification omics and other techniques.In this study,we established a system to automatically identify individual cells within micrographs and track their trajectories to improve efficiency for subsequent batch analysis of data.The image recognition part of the system is composed of MaskRCNN image recognition neural network,which can accurately and quickly identify single cells after a large number of training.The trajectory tracking part is composed of the python language plug-in package Usiigaci,which can trace the data obtained by identifying a single cell,and integrate the tracking coordinate data for output and graphical display.Subsequently,Excel was used to transform the data,and the output coordinate data were further transformed into visual charts through the formula group.The transcriptome data analysis of exogenous electric field and non-electric field showed that the expression of most Ras protein families in Dictyostelium cells stimulated by electric field was significantly upregulated.Targeted proteomics showed that phosphorylation levels of some Ras GEF(gef)family proteins were upregulated.Among them,Ras family proteins and Ras GEF(gef)family proteins are both upstream pathways of MAPKs pathway,and it is speculated that their interaction ultimately affects cell electrotaxis.Therefore,four Ras GEF family knockout mutants,gef D null,gef U null,gef T null and PaxB null,were generated by screening the rasge F family knockout strains from the Dictyostalt mutant library.The gef D null mutant strain was selected for electrotaxis experiment,which showed that the gef D protein deletion would lead to the reduction of cell migration speed and direction.Moreover,Ras proteins that may interact with gef D in vitro were identified by yeast two-hybrid assay,and Ras D protein was the most closely interacted with gef D.The gef T null and gef U null knockout mutants were subjected to developmental experiments,electrotaxis experiments,cell polarization experiments,actin fluorescence localization and Western blot experiments,respectively.The results of cell electrotaxis experiment showed that the polarization of Gbp C Ras GEF domain knockout mutant(gef T null)disappeared,but the cell electrotaxis remained unchanged.The Ras GEF domain knockout mutant(gef U null)was hyperpolarized,but the electrotaxis of the cells were significantly reduced.Western blotting showed that the myosin light chain of gef T null mutant underwent rapid dephosphorylation under electric field stimulation and recovered gradually over time.However,the phosphorylation level of myosin light chain of gef U null mutant decreased gradually under electric field stimulation.However,the development process of PaxB null mutant strain was severely blocked and could hardly complete the final development process.Cell polarization assay showed that the polarization phenotype of PaxB null mutant was the same as that of the wild type,but its migration rate was higher than that of the wild type.Cell electrotaxis showed that PaxB null mutant migrated faster than the wild type,but had lower directivity.Western blotting showed that the phosphorylation level of ERK1/2 of PaxB null mutant was significantly higher than that of the wild type,but the phosphorylation level of ERk1/2of PaxB Null mutant was not affected by electric field stimulation.